Tendinopathies are common musculoskeletal injuries that often lead to tendon rupture and loss of function. An increase in glycosaminoglycan (GAGs) content is one of the most prominent characteristics of late stage tendinopathy. Despite the prevalence of this observation in clinical patients, a fundamental question still remains: is the increase in GAGs that is characteristic of late stage tendinopathy a result of the altered cellular loading environment or part of the mechanistic pathway by which cells modulate the altered loading environment to maintain survival and homeostasis? Answering this question is critical, as it would inform whether the goal for effective interventions should aim to increase or decrease GAG content post sub-rupture fatigue damage injury in order to halt or reverse the progression of damage. Interestingly, using our established in vivo model of sub- rupture damage accumulation in the patellar tendon (PT) to investigate the onset and pathogenesis of tendinopathy, we have found that the increase in GAGs, particularly dermatan sulfate (DS) and hyaluronan (HA), that is associated with late stage tendinopathy initiates after just 1 bout of fatigue loading. We have also found that physiological exercise that is initiated prior to the increase in DS and HA leads to further degeneration, but exercise that is initiated after the increase in DS and HA promotes remodeling. These findings highlight the potential contribution of GAGs to the capacity of the tendon to repair and suggests that investigating their role in our model will ultimately motivate diagnostics to guide clinical management of fatigue damaged tendons. Accordingly, this proposal will test the hypothesis that the early increase in DS and HA modulates the mechanical environment of the resident cells to restore interactions with the extracellular matrix (ECM) and promote survival; a mechanistic necessity for the fatigue damaged tendon to repair from subsequent therapeutic loading. We will determine the effect of the increase in DS and HA post fatigue loading on (1) multi-scale tendon mechanics and the mechanical environment of the cells (Aim 1); and (2) promotion of cell survival, maintenance of cell phenotype, and modulation of the biological environment that may be necessary for an effective response to therapeutics after fatigue injury (Aim 2). We will utilize our fatigue damage model in combination with novel imaging applications and visualization of deformations of GFP+ cells with post mortem (Aim 1) and in vivo (Aim 2) digestion of HA and DS. The proposed studies will propel progress in the field by providing goals for therapeutics regarding modulation of GAGs in fatigue damaged tendons. In addition, these studies will be foundational to establishment of diagnostic tools to guide clinicians as to whether a patient will benefit from exercising a fatigue damaged tendon or will incur further injury; a major step towards advancement of patient specific management of sub-rupture tendon injuries.